Surface-modified polyacrylonitrile beads

ABSTRACT

A bead composition is disclosed herein which comprises a core of a polyacrylonitrile homopolymer or copolymer and a surface of pendant N-haloamide groups. Also disclosed is a process for the production of said composition.

This is a divisional of co-pending application, Ser. No. 07/551,597,filed Jul. 11, 1992, U.S. Pat. No. 5,137,982 which in turn, is acontinuation-in-part Ser. No. 07/348,448, filed May 8, 1989, nowabandoned which is a continuation-in-part of Ser. No. 07/349,569 filedMay 18, 1989, now abandoned.

This application is related to commonly assigned patent applicationsentitled "Surface-Modified Polyacrylonitrile Beads" Ser. No. 07/349,569and "Surface-Modified Polyacrylonitrile Fibrous Substrates" Ser. No.07/348,454, both of Laurence Wu-Kwang Chang, Larry Stanley Anderson andDavid Arthur Ley.

This application is further related to commonly assigned patentapplications of Michael Timothy Cooke and Laura Jean Hiscock for PorousPolyacrylonitrile Beads and Process, Ser. No. 07/275,317, MichaelTimothy Cooke and Laura Jean Hiscock, for Porous Polymer Beads andProcess, Ser. No. 07/275,256, and David Arthur Ley, Laura Jean Hiscockand Michael Timothy Cooke for Process for the Preparation of PorousPolymer Beads, Ser. No. 07/275,170, as well as Michael Timothy Cooke,Larry Stanley Anderson and David Arthur Ley for Surface-hydrated PorousPolyacrylonitrile Substrates, Such as Beads, Derivatives Thereof,Processes for their Preparation and Methods for their Use, Ser. No.07/276,183. The contents of these applications are hereby incorporatedby reference.

This invention relates to substrates having a core comprising anacrylonitrile polymer or copolymer thereof and N-haloamide groups on thesurface thereof; and a process for their production. One embodiment ofthe present invention is directed to porous isotropic beads ofpolyacrylonitrile or a copolymer thereof having N-haloamide groupspendant on the surface thereof.

This invention further relates to substrates wherein the aforementionedpendant N-haloamide groups form functionalized substrates. Saidfunctionalized substrates may be used to form complexes with biologicalsubstances, thereby permitting, for example, the separation of saidsubstances from solutions in which they are contained.

Substrates produced in accordance with the present invention are usefulin various applications including chromatography separation processes.

BACKGROUND OF THE INVENTION

Rigid non-swellable polymeric materials having neutral, hydrophilicsurfaces are useful for many applications. These include chromatographysupports, membranes, carriers for immobilized enzymes or immunoassaysupports. Hydration of polyacrylonitrile surfaces to form acrylamidegroups is well known in the art.

U.S. Pat. No. 4,110,529 (Stoy), discloses the introduction of reactivegroups into the surface layer of beads during coagulation. Example 5 ofthe Stoy patent discloses the partial hydration of a polyacrylonitrileto 40 percent amide groups and then coagulation to form porous beads.However, beads prepared in this manner are highly swellable in water andcontain a substantial amount of byproduct carboxylate groups in additionto the desired amide groups. Thus, the beads are not particularly usefulas chromatographic supports. Their tendency to swell results inexcessive pressure drops and inconsistent flow rates in chromatographiccolumns and the presence of carboxylate groups causes non-specificbinding in separation processes not involving ion exchange. Problemsalso arise from the high (up to 40%) amide conversion rate as highconversion to amide groups results in significant losses in beadstrength and chromatographic flow due to loss of bead rigidity.

Other attempts to convert nitrile groups to amides in the prior art haveinvolved treatment with strong acids or bases. However, both of thesetechniques generally lead to some formation of surface carboxyl groups.For example, U.S. Pat. No. 4,143,203 (Rigopolous) discloses solidparticles possessing an impermeable rigid polyacrylonitrile core with ahydrated surface. The surface is hydrated by heating the solidpolyacrylonitrile particles in a solution of sulfuric acid attemperatures ranging from 75° to 95° C. However, the beads formed underthese conditions are non-porous and also contain a substantial amount ofbyproduct carboxyl groups. They are therefore not useful in non-ionexchange protein specific chromatographic applications.

The surface modification of polyacrylonitrile under basic conditions wasstudied by K. Ohta et al., Nippon Kagaku Kaishi, 6. 1200 (1985) usingsurface infrared spectroscopy. After treating polyacrylonitrile filmswith 5 percent sodium hydroxide solution for 4 hours at 70° C., Ohtareported finding 4.5 percent amide and 5.7 percent carboxylate groups onthe surface of the film. Treatment of the film with a solution of 5percent sodium hydroxide and 15 percent hydrogen peroxide (an aqueousalkaline peroxide reaction) for 4 hours at 70° C. reportedly produced2.1 percent amide and 0.7 percent carboxylate. These treatments aretherefore not sufficiently selective.

Thus, until recently the state of the art still encountered seriousdrawbacks to the formation of highly selective non-swellable highlyporous acrylonitrile substrates having neutral hydrophilic surfaces. Thegreater surface area of highly porous beads and the narrow diameter ofthe polymer structure, makes it critical to accurately control theextent of hydration. Conversion of more than 15 percent of the nitrilegroups to amide groups results in significant losses in flow inchromatography separations. It is difficult to accurately control theextent of reaction with acidic hydration. Acidic hydration is also knownto have a strong neighboring group effect which generates a "block"polymer structure. A block polymer structure at low conversion canresult in non-uniform coverage of the surface. Again, this causesproblems with non-specific binding in chromatography applications. Athird problem with acidic hydration is the formation of carboxyl andimide groups. The presence of carboxyl groups as previously statedcauses undesired ion interactions during size exclusion or affinitychromatography applications.

It has been disclosed in commonly assigned application Ser. No.07/276,183 that alkaline peroxide hydration of nitriles, with carefulcontrol of the solvent utilized, can avoid the aforementioned problems.The reaction selectively converts nitrile groups to amide groups withoutside reactions to imide or carboxyl groups. By proper selection of thesolvent, the reaction can be easily controlled and actually stopped atlow conversion. The use of solvent, preferably methanol, allows all ofthe surfaces of the substrate (as hereinafter defined), to be converted.The process disclosed therein produces an even distribution of amidegroups on the surface of the substrate.

The rigid nature of the polyacrylonitrile core is minimally effected bythis mild treatment and thus, the substrates are non-compressible andsubstantially non-swellable in water. When used therein, the term"non-compressible" denoted the resistance to hydrostatic pressures incolumnar beds of up to about 3000 psi. without collapsing to preventflow therethrough.

A method has now been found to convert substrates, such as the surfacetreated substrates disclosed in above-discussed U.S. Ser. No.07/276,183, such that said substrate bears pendant N-chloroamide groupson the surface thereof while the core of the substrate remainsunreacted. The substrates so produced are useful as intermediates in theproduction of various surface treated products which bear functionalmoieties linked to the core of the substrate through reaction of thependant N-chloroamide group.

SUMMARY OF THE INVENTION

In accordance with the present invention there are provided substratescomprising:

a) a core comprising polyacrylonitrile, or a copolymer of acrylonitrileand at least one comonomer, and

b) a surface having evenly distributed thereon N-chloroamide groups and,optionally, nitrile and amide groups.

In a preferred embodiment of the present invention, there is provided asubstantially skinless porous bead having a pore-volume of notsubstantially less than 1.5 ml/g and being substantially isotropic,comprising a core of polyacrylonitrile, or a copolymer thereof, andamide and N-chloroamide groups, and optionally, nitrile groups, evenlydistributed over the surface thereof.

Also in accordance with the present invention there is provided aprocess for the preparation of the aforementioned substrates saidprocess comprising

a) contacting a substrate comprising polyacrylonitrile, or a copolymerof acrylonitrile and at least one comonomer with an alkaline catalyst, aperoxide, and optionally a reducing agent, under reaction conditions andfor a time sufficient to convert at least a portion of the nitrilegroups distributed on the surface of the substrate to amide groups;

b) contacting said substrate with a halogenating reagent under reactionconditions and for a time sufficient to convert at least a portion ofthe amide groups to N-haloamide groups; and

c) recovering the surface-modified substrate.

Also disclosed herein is a process for the preparation of theaforementioned polyacrylonitrile porous bead substrates, said processcomprising

a) forming a suspension in a liquid non-solvent for the polymer orcopolymer beads comprising polyacrylonitrile or a copolymer ofacrylonitrile and at least one comonomer;

b) adding an alkaline catalyst, a peroxide and optionally a reducingagent to said suspension and heating for a time sufficient to convert upto about 15 mole percent of the total nitrile groups through hydrationto amide groups;

c) recovering said beads from said suspension;

d) contacting said beads with a halogenating reagent under conditionsand for a time sufficient to convert at least a portion of said surfaceamide groups to N-haloamide groups; and

e) recovering the surface-modified porous polymer beads.

Also disclosed herein is a composition of matter useful in the recoveryand/or isolation of biological material, said composition of mattercomprising

a) a core comprising polyacrylonitrile, or a copolymer of acrylonitrileand at least one comonomer, and

b) a surface having evenly distributed thereon,

i) pendant bioactive ligand groups, said bioactive ligand groups beingbound to said surface through linkages derived from bioactive ligandsand N-haloamide groups bound to said surface, and optionally,

ii) nitrile and/or amide groups.

Further disclosed herein is a process for the production of saidcompositions of matter useful in the recovery and/or isolation ofbiological material, said process comprising

a) reacting a substrate comprising polyacrylonitrile or a copolymer ofacrylonitrile and at least one comonomer with an alkaline catalyst, aperoxide and optionally a reducing agent under reaction conditions andfor a time sufficient to convert at least a portion of the nitrilegroups distributed on the surface of the substrate to amide groups;

b) reacting said substrate with a halogenating agent under conditionsand for a time sufficient to convert at least a portion of said surfaceamide groups to N-haloamide groups;

c) reacting said substrate with a bioactive ligand such that saidbioactive ligand is bound to said substrate through a linkage comprisingsaid N-haloamide groups; and

d) recovering said substrate.

Also disclosed herein are processes for the recovery and/or isolation ofbiological material using the above-identified compositions of matter.

DETAILED DESCRIPTION OF THE INVENTION

Substrates comprising polyacrylonitrile homopolymers or copolymers aregenerally known. For instance, semi-permeable membranes ofpolyacrylonitrile are utilized in various chemical separations. Hollowfibers of polyacrylonitrile, such as those marketed by Asahi MedicalCompany Ltd. under the designation PAN 140, are currently used in kidneydialysis equipment.

Porous bead substrates comprising acrylonitrile polymers or copolymersare known to those skilled in the art and utilizable in the practice ofthe present invention. One method for preparing porous copolymers isdescribed in U.S. Pat. No. 4,246,351. A preferred method of preparingporous polyacrylonitrile beads is disclosed in the above-mentionedcommonly assigned copending U.S. patent applications, Cooke and Hiscock,Ser. No. 07/275,317, Ley, Hiscock, and Cooke, Ser. No. 07/275,170, andCooke and Hiscock, Ser. No. 07/275,256, all of which are filed onNovember 23, 1988. The thermally induced phase separation processdisclosed therein provides microporous beads comprising acrylonitrilepolymers or copolymers thereof which are substantially skinless,isotropic, and have a high pore volume. Such porous bead substrates areamong the preferred substrates used in the practice of the presentinvention. Also preferred are polyacrylonitrile substrates suchnon-porous sheets or films, porous membranes, hollow fibers includingporous fibers, monofilaments, acrylic yarns and fibrillated fibers. Itshould be readily apparent that the form of the substrate is notcritical to the practice of the invention disclosed herein.

As mentioned above, the polyacrylonitrile substrates may compriseacrylonitrile homopolymers or copolymers. Suitable comonomers compriseC₂ -C₆ mono-olefins, vinyl aminoaromatics, alkenyl aromatics, vinylaromatics, vinyl halides, C₁ -C₆ alkyl(meth)acrylates, acrylamides,methacrylamides, vinyl pyrrolidones, vinyl pyridine, C₁ -C₆hydroxyesters of alkyl(meth)acrylates, meth(acrylic)acids,acrylomethylpropylsulfonic acids, N-hydroxy-containing C₁ -C₆alkyl(meth)acrylamide, acrylamidomethylpropylsulfonic acids, vinylacetate, glycidyl (meth)acrylate, glycerol (meth)acrylate,tris(hydroxymethyl) amino methyl (meth)acrylamide or a mixture thereof.Acrylonitrile copolymers may comprise from about 99 to about 20 parts byweight acrylonitrile and from about 1 to about 80 parts by weightcomonomer. It is preferable that the acrylonitrile be present in greaterthan about 90 mole percent and the preferred comonomer comprises methylacrylate.

As used herein in the physical description of the substrates of thepresent invention, the term "surface" means the interface of thesubstrate and non-substrate. The term "core" as used herein denotes thatportion of the substrate other than its "surface".

In the practice of the present invention, the surface of theacrylonitrile substrates is initially hydrated such that the surfacebears pendant amide groups. This is accomplished by reacting at least aportion of the nitrile groups present on the surface of the substratewith an alkaline peroxide and optionally a reducing agent in a liquidnon-solvent for the polymer. The reaction selectively hydrates nitrilegroups to amide groups without side reactions to imide or carboxylgroups. The product of this reaction wherein at least a portion of thesurface nitrile groups have been converted to amide group is hereinafterreferred to as "Substrate I". Furthermore, this initial reaction of thepresent invention is surprisingly easily controlled and conversions ofless than about 15 mole percent nitrile groups to amide groups arereadily obtainable. This reaction is generally disclosed in commonlyassigned patent application Ser. No. 07/276,183 which is referred toabove.

If beads are utilized as the substrate, the process to produce SubstrateI comprises forming a suspension of the beads and a non-solvent for thepolymer comprising said beads and the pendant polyacrylamide surfacewhich is formed. It is also contemplated to introduce a catalyst intothe suspension. The suspension is then stirred and an alkaline reagentadded. The suspension is then heated, a peroxide and preferrably areducing agent is added while the suspension is stirred, and thereaction is carried out to the desired extent.

Suitable peroxides for use in the practice of the present inventioninclude hydrogen peroxide, t-butyl hydroperoxide, or mixtures thereof,and the like. Especially preferred is hydrogen peroxide.

Many alkaline reagents are known to those skilled in the art and aresuitable for use in this invention. Alkaline reagents include sodiumhydroxide, potassium hydroxide, or mixtures thereof, and the like.

The reducing agent can be any agent capable of reacting with theintermediate hydroperoxide. Especially preferred is dimethyl sulfoxide.

Essential to the practice of the present invention is the choice of asuitable solvent for the reaction. The choice and concentration of thecomponents of the solvent system is believed to control the selectivityand extent of the reaction. Although applicants do not wish to be boundby any theory, it is believed that the ability of the solvent system todissolve the alkaline reagent and peroxide while having only limitedability to solvate the amide groups as they are formed controls theextent of the reaction. Thus by controlling the ratio of the solventcomponents, the extent of the reaction can be controlled. Preferably,where hydrogen peroxide is the peroxide utilized and dimethyl sulfoxideis the reducing agent, sodium hydroxide is the alkaline reagentutilized, methanol is employed as solvent with limited ability tosolvate the polyacrylacrylamide surface as it is formed.

Following the initial reaction wherein surface nitrile groups areselectively converted to amide groups, Substrate I is then removed fromcontact with the reagents used in its production. It is preferred thatthe substrate has at least 2.5% of the nitrile groups found on thesubstrate surface converted to amide groups. Most preferably, in poroussubstrates about 10-15% of the total nitrile groups are converted toamide groups.

If porous beads are utilized as the substrate, it is preferred that thebeads are annealed prior to their introduction into the suspension withthe non-solvent. The annealing step is most preferably carried out asfollows: The beads are dried at a temperature of less than 50° C. andthen heated to 90°-100° C. for a period ranging from about 30 to about60 minutes. The annealing appears to decrease the reactivity of thebead. Although applicants do not wish to be bound by any single theory,it is postulated that this decrease in bead reactivity occurs by thepolymer becoming more ordered and/or decreasing the surface area.Annealing, thus affects the nitrile to amide ratio and should thereforebe anticipated in the practice of the present invention.

Substrate I is then subjected to a second reaction such that at least aportion of said amide groups are converted to N-haloamide groups. Apreferred embodiment are the formation of N-chloroamide groups. It isimportant that this conversion be accomplished without the generation ofby-products through reaction of the pendant nitrile groups or otherunacceptable degradation of the substrate. In the selection of thesolvent for the halogenation reaction, the solvent must be inert to thehalogenating agent and a non-solvent for the polymer comprising thesubstrate. Preferred solvents include water, carbon tetrachloride,tetrachloroethane and chlorobenzene. The halogenation reaction involvesthe use of the amide-bearing substrate with halogenating agents, such aschlorine gas, t-butyl hypochlorite, chloromonomide, sodium hypochlorite,hypochlorous acid, sodium hypobromite, and mixtures thereof. Typically,the halogenating agents are present in solution in amounts ranging fromabout 1.0 eq. to about 2.0 eq. based on the amide content of thesubstrate. Using the formation of N-chloroamides as an example, thechlorinating agents are contacted with Substrate I for sufficientperiods to affect the chlorination of the desired portion of the surfaceamide groups. Typically, contact times range from about 0.5 hours toabout 4.0 hours, preferably from about 1 hour to about 3 hours whenabout 1.25 eq. solutions are employed. The temperature at which thechlorination reaction is conducted is not critical. Typical reactiontemperatures range from about 0° to about 40° C., preferably from about10° to about 30° C.

Conversion of pendant amide groups on surface of Substrate I toN-chloroamide groups may be controlled by limiting the reaction time andthe concentration of the chlorinating agent. Preferably, conversions of25% to about 100% may be accomplished while 50-90% conversion rates aremore typically encountered. For porous substrates, an N-chloroamidecontent of about 0.5-3.0 mMole/gram is preferred while those possessinga content of about 1.0-2.0 on the identical basis are especiallypreferred.

Following completion of the reaction to the desired level ofhalogenation, the substrate (which shall hereinafter be referred to as"Substrate II") is removed from contact with the halogenating agent.Optionally, the substrate may be washed to remove remaining quantitiesof the halogenating agent. Although optional, this step is particularlydesirable when porous substrates are used due to their tendency toretain the halogenating agent in their pores.

Substrate II, produced through the above-described reaction comprises ahomopolymeric or copolymeric acrylonitrile core and a surface bearingN-haloamide groups. Optionally and directly dependent upon the degree towhich the preceding reactions were carried out, the surface of SubstrateII may further bear amide nitrile and the comonomer groups that have notundergone reaction.

Substrate II is particularly well suited, due to the presence of thependant N-haloamide groups, to use as an intermediate useful in theproduction of various final products since the chemistry associated withN-haloamide reactions is widely known. N-haloamides are well known toundergo a Hofmann rearrangement to form isocyanates. For instance, U.S.Pat. Nos. 4,301,257, 4,356,289 and 4,357,447 disclose the production ofsoluble polymeric isocyanates from N-chloroamides. A general discussionof Hofmann rearrangements is further contained in J. March AdvancedOrganic Chemistry: Reactions, Mechanisms and Structure pp 816-817 McGrawHill, Inc. 1968. For example, Chemical Reviews, Volume 72, pp. 457-496(1972) discusses various reaction schemes which could be employed in theproduction of numerous products from the isocyanate intermediate formedfrom Substrate II. For instance, products useful in affinitychromatography, dye affinity chromatography, metal ion affinity, ionexchange, hydrophobic interactions and reverse phase chromatography canbe so produced.

Of particular interest is the production of products useful in the areaof bioseparations and/or affinity chromatography. These products(hereinafter referred to as "Biosubstrates") are produced through theattachment to Substrate II through the pendant surface N-haloamidegroups of functional groups (bioligand) which are capable of bindingwith biological material. The term "binding" as used herein is to beinterpreted broadly to encompass not only covalent bonding but also allless powerful interactions, such as electrostatic forces, van der Waalsforces, and hydrogen bonding.

Production of Biosubstrates through attachment of bioligands toSubstrate II may be accomplished directly or through the use of anintermediate bridging group which may facilitate such attachment. Forinstance, bioligands having pendant H₂ N--, HO-- or HS-- groups mayattach directly to Substrate II. Therefore a cationic exchange resinbearing pendant groups ##STR1## may be produced through the directreaction of Substrate II with H₂ N--R--CO₂ H where R is, for example, aC₁ -C₁₈ alkylane group.

Methods of binding bioligands to various substrates and theiractivation, if necessary, are generally known. For example, thefollowing references contain such disclosure, the contents of which arehereby incorporated by reference.

J. Turkova, Affinity Chromatography, Journal of Chromatography Library,Elsevier, Vol. 12 pp. 151-202 (1978).

L. Jervis, Syntheses and Separations using Functional Polymers, ed. D.C. Sherrington and P. Hodger, John Wiley and Sons Ltd., pp. 265-304(1988).

Bioligands, as noted above, are chemical and biological moieties capableof binding with biological materials. The term bioligands, as usedherein, also includes moieties which are capable of binding withbiological material subsequent to their activation. For example,bioligands having pendant groups, such as --CO₂ H, --SO₃ H, --NR₂ or--NR₃ where R is a C₁₋₆ alkyl group, are capable of binding withbiological materials by an ion exchange mode. Bioligands having C₁ -C₁₈alkyl can bind with biological materials by hydrophobic interactions.

In another preferred embodiment, hydrazide functionality can be attachedto the surface of the substrates. Hydrazides can couple bioactiveligands either after activation with nitrous acid ( J. Turkova, AffinityChromatography, Journal of Chromatography Library, Vol 12, p. 175, 1978,Elsivier Science Publishers, B.V.) or by site specific reaction with theoxidized carbohydrate portion of immunoglobumins (W. L. Hoffman and D.J. O'Shannessy, J. Immunological Methods, 112, p. 113-120 (1988)).Hydrazide functionality can be introduced by a variety of syntheticroutes which can adjust the length of the side-arm. In a preferredmethod, the N-chloroamide surface can be reacted with either malonicdihydrazide or adipic dihydrazide to produce the hydrazidefunctionalized surface.

Several alternative methods for creating other spacers arms among whichare: reaction of hydroxyl functional surfaces of the present inventionwith methyl fhloroformate followed by hydrazide; reaction of aldehydefunctional surfaces with either malonic or adipic dihydrazide followedby reduction of the Schiff base; or reaction of a primary aminefunctional surface with succinic anhydride followed by esterificationand reaction with hydrazine.

Strongly acidic and basic ion exchange surfaces cal also be prepared.Introduction of sulfonic acid groups may be accomplished by reaction ofthe N-chloroamide surface with a sulfonic acid-containing compound suchas 2-aminoethane sulfonic acid. Quaternary ammonium surfaces can beprepared by quaternization of the tertiary amine surfaces. Alkyl halidesor epichlorohydrin are convenient reagents for this reaction.

Alternately, Biosubstrates may be produced wherein the bioligand isbound to Substrate II by way of a bridging group. This is a preferredmode for attachment of bioligands derived from biological material. Theidentity of the bridging group is not critical in the practice of thepresent invention. However, it must be at least difunctional and becapable of reaction with both the pendant N-haloamide groups ofSubstrate II and a bioligand without unduly degrading either or undulyinterfering with the performance of the Biosubstrate produced. Bridginggroups may consist of various difunctional compounds includingpolyalkylene glycols, such as polyethylene glycols and polypropyleneglycols, preferably those having low molecular weights from 62 to 250;monosacharides, such as fructose, glucose, mannose, ribose, galactose;disaccharides, such as sucrose, maltose, lactose, cellobiose, diamines,such as ethylene diamine, hexamethylene diamine, 1,3-diamino-2-propanol,amino acids, such as glycine, beta-alanine, 6-aminocaproic acid;acyldihydrazides, such as succinic dihydrazide, adipic dihydrazide. Thebridging group may have any of the usual chain lengths, being made fromdifunctional compounds having chain lengths from one to 15 or more atomsbetween the two reactive functional groups. Bridging groups bearingpendant groups, such as --ROH, --RCO₂ H, --RNH₂, RCHO, ##STR2## requireactivation and/or reaction with other bioligands prior to their use asBiosubstrates. A bioligand preferably employed in the practice of thepresent invention is Protein A.

Bridging groups and their use in the production of Biosubstrates aregenerally known. For example, the Turkova and Jervis references notedabove disclose their usage. Typically, bridging groups comprisealiphatic, aromatic or cycloaliphatic hydrocarbonaceous groups. They mayoptionally contain heteroatoms, such as O, N or S. Further, the bridginggroups typically contain from 1 to about 15 carbon atoms.

In the practice of the present invention, the bridging groups arepreferably derived from the following reactants: mono-, di- ortrialkylene glycols, alkylene mono-, di- or trialkylene amines, lowerdiols and polyols, alkanolamines and amino acids. Particularly preferredreactants used in the derivation of a bridging group between a bioligandand Substrate II include ethylene glycol, diethylene glycol, triethyleneglycol, glycerol, ethylenediamine, diethylenetriamine, ethanolamine,diethanolamine, 3,3'-diamino-N-methylpropylamine, hexanediamine,glycine, beta-alanine, tris(hydroxymethyl)aminomethane, 6-aminocaproicacid and polyoxyethylenediamine.

Bridging groups can also be formed through sequential reactions in thepresence of Substrate II or they may be preformed prior to the reactionwith Substrate II.

Particularly preferred as bridging groups in the production ofBiosubstrates in the practice of the present invention are the reactionproducts of:

diethylene glycol followed by its reaction with2-fluoro-1-methylpyridinium-p-toluenesulfonate, and

2) alkylenediamine and succinic anhydride followed by its reaction withN-hydroxysuccinimide, and

3) 6-aminocaproic acid followed by its reaction withN-hydroxysuccinimide.

Following the reaction of Substrate II with a reactant to generate abridging group or Biosubstrate, remaining N-haloamide functionalitiespresent on Substrate II are typically destroyed, preferably such thatless than about 0.1 mMole/gram of active chlorine remains. This may beaccomplished through use of heat, treatment with a reducing agent (suchas sodium sulfite) or both.

Once produced, Biosubstrates may be utilized in applications where theiraffinity for and ability to bind with biological material can beutilized. For example, Biosubstrates can be used to isolate biologicalmaterial from solutions in which it is contained by adding theBiosubstrates to said solutions or by passing said solution over a fixedbed of the Biosubstrate. The Biosubstrate is then isolated from thesolution. Optionally, the Biosubstrates can be separated from saidsolution of biological material to allow for the isolation of thebiological material and recycle or reuse of the Biosubstrate.

EXAMPLES

The following Examples are presented to illustrate the practice of thepresent invention. They should not be construed however as limitationsof the scope of the present invention.

PROCEDURE A

Five grams of a wet copolymer containing 99 mole percent acrylonitrileand mole percent of methyl acrylate (1:1 copolymer:water by weight) wereground with 5 grams of urea and 30 grams of dimethylsulfone to form apowdered mixture. The mixture was placed in a 1 liter flask containing100 ml of mineral oil heated to 160° C. The mixture was stirred untiltwo liquid phases were present, one phase being a homogeneous polymersolution, the other mineral oil. Rapid stirring of the mixture with anoverhead paddle stirrer gave a suspension consisting of droplets of thehot (about 120° C.) polymer solution in mineral oil. The droplets werecooled by transferring the suspension via a canula to a second stirredmixture consisting of 500 ml of mineral oil, 6 grams of dimethylsulfone,and 1 gram of urea kept at 70° C. The droplets solidified uponcontacting the cooler mineral oil. The mixture was cooled with stirringto room temperature, then diluted with methylene chloride to reduce theviscosity of the oil. The droplets were collected on a Buchner funneland washed with methylene chloride, then the solvent was extracted with200 ml of acetone for 1.5 hours at room temperature. The resulting beadswere examined by scanning electron microscopy and seen to be highlyporous, with relatively uniform pore diameter of about 0.5 microns. Thepores extended through the outer surfaces of the beads. The beads rangedin size from 10 microns to a few millimeters in diameter.

Another detailed example of preparing these porous polymer beads is asfollows:

Two-hundred eighty-eight grams of dimethylsulfone, 12 grams ofacrylonitrile copolymer consisting of a 99:1 mole ratio acrylonitrile:methyl acrylate, and 100 ml of propylene glycol were combined and placedin a Parr reactor equipped with a magnetically driven stirrer and dipleg. The reactor was heated to 140° C. to form a homogeneous solution.The solution was forced through heated (140° C.) lines and anatomization nozzle (Lechler Co. full cone "center jet" nozzle, 0.46 in.diameter orifice) using 150 psig nitrogen pressure. The nozzle wasmounted 3 inches over 3 liters of stirred mineral oil or 4 inches over 4liters of stirred heptane to quench the liquid droplets. The solidifieddroplets were washed with heptane to remove mineral oil, dried andextracted for one hour with 3 liters of 85°-90° C. water to producemicroporous beads. Pore sizes ranged from 0.05 to 1.5 microns and themajority of the beads are between 25 and 150 microns.

The following examples illustrate the production of Substrate I whichbears pendant amide groups.

EXAMPLE 1A

A suspension of 5 grams of dry annealed polyacrylonitrile beads (45-90microns, 94.5 mmoles) in 115 ml of methanol, 5 ml of water, and 4 ml ofdimethylsulfoxide (56.4 mmoles) were stirred under a nitrogen purge.After ten minutes of purging, 2.4 ml of 2N aqueous sodium hydroxide (4.8mmoles) were added to the suspension and the suspension heated to 35° C.Hydrogen peroxide, 4.9 ml of a 30 percent solution (47.9 mmoles) wasadded over 10 minutes. The reaction mixture was stirred at 35° C. forthree hours. After 3 hours, 2.4 ml of 2N hydrochloric acid (4.8 mmoles)was added and the reaction mixture was stirred for one minute andfiltered. The beads were washed with 0.1N aqueous hydrochloric acid,water, methanol and then dried. The amide content of the beads wasdetermined to be 9.7 percent by infrared analysis.

The following Examples illustrate the production of Substrate I whichbears pendant amide groups as well as Biosubstrates made therefrom.

EXAMPLE 1B

Dry annealed polyacrylonitrile hollow fibers, 0.5 g, were mixed with11.5 ml of methanol, 0.5 g of water, 0.24 ml of 2N aqueous sodiumhydroxide solution, and 0.4 ml of dimethyl sulfoxide. The mixture washeated to 35° C., and 0.49 ml of a 30% hydrogen peroxide solution wasadded. After standing at room temperature for 3 hrs., the reactionmixture was filtered. The fibers were washed with water and methanol andvacuum dried (40° C.). The amide content of the fibers was determined tobe 14.1% by infrared analysis.

EXAMPLE 1C

The procedure of Example IB was followed except that 0.50 g of anon-annealed fibrillated fiber sheet was used with 1.47 ml of 30 percenthydrogen peroxide solution was used, and the fiber was annealed beforethe reaction. IR analysis showed that the amide content of the MAPfibrillated fiber product was about 2%.

EXAMPLE 1D

The reaction procedure of Example IB was followed except that 0.52 g ofa nonporous film made from a 89.5: 10.5 acrylonitrile:methyl acrylatefilm was used and the film was not annealed. Contact angle for water was42°; initial film had a water contact angle of 63°.

The following Examples illustrate the production of Substrate II whichbears pendant N-chloroamide groups as well as the Biosubstrates madetherefrom.

EXAMPLE 2

The product of Example 1A, 3 g, was mixed with 78 ml of water. To thissuspension was added 0.471 g of chlorine gas. The addition time of thechlorine was 11 minutes. The reaction mixture was stirred at roomtemperature for 2 hrs. After 2 hrs. the reaction mixture was filtered.The beads were washed with water and then vacuum dried (40° C.).Iodometric titration showed that the beads contained 1.40 mmole/g ofactive chlorine corresponding to ca. 80% chlorination of the amidegroups on the beads.

EXAMPLE 3

A solution of 130 ml of diethylene glycol (DEG) and 6.3 ml of 2N aq.sodium hydroxide was heated to 40° C. To this solution was added 5 g ofthe product of Example 2. The reaction mixture was stirred at ca. 40° C.for 2 hrs. After two hrs., the reaction mixture was filtered. The beadswere washed with water and then vacuum dried (40° C.).

Infrared spectroscopy confirmed that DEG had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 4

The procedure of Example 3 was followed, except that 100 ml of ethyleneglycol (EG), 3.3 ml of 2N aq. sodium hydroxide and 2 g of the product ofExample 2 were used for the reaction.

Infrared spectroscopy confirmed that EG had been reacted with said beadsthrough a Hofmann rearrangement.

EXAMPLE 5

The procedure of Example 3 was followed, except that 235 ml oftriethylene glycol (TEG), 3.8 ml of 2N aq. sodium hydroxide and 3.5 g ofthe product of Example 2 were used.

Infrared spectroscopy confirmed that TEG had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 6

The procedure of Example 3 was followed except that 100 ml of methanol,2.3 ml of 2N aq. sodium hydroxide and 2 g of the product of Example 2were used.

Infrared spectroscopy confirmed that methanol had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 7

The procedure of Example 3 was followed except that 65 ml of glycerol,2.3 ml of 2N aq. sodium hydroxide, and 2 g of the product of Example 2were used, and 4 ml of water was added to the glycerol solution prior tothe addition of the beads.

Infrared spectroscopy confirmed that glycerol had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 8

A solution of 90 ml of ethylenediamine (EDA) and 3 ml of water washeated to 40° C. To this solution was added 2 g of the product ofExample 2. The reaction mixture was stirred at 40° C. for 2 hrs. Aftertwo hrs., the mixture was filtered. The beads were washed with water andthen vacuum dried (40° C.).

Infrared spectroscopy confirmed that EDA had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 9

The procedure of Example 8 was followed except that 100 ml ofdiethylenetriamine (DETA), 3 ml of water, and 2 g of the product ofExample 2 were used.

Infrared spectroscopy confirmed that DETA had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 10

The procedure of Example 8 was followed except that 100 ml ofethanolamine (EA), 3 ml of water, and 2 g of the product of Example 2were used.

Infrared spectroscopy confirmed that EA had been reacted with said beadsthrough a Hofmann rearrangement.

EXAMPLE 11

The procedure of Example 8 was followed except that 75 ml ofdiethanolamine (DEA), 50 ml of water, and 2 g of the product of Example2 were used.

Infrared spectroscopy confirmed that DEA had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 12

The procedure of Example 8 was followed except that 75 ml oftriethanolamine (TEA), 50 ml of water, and 2 g of the product of Example2 were used.

Infrared spectroscopy confirmed that TEA had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 13

The procedure of Example 8 was followed except that 98 ml of propylamine(PA), 2.5 ml of water, and 2 g of the product of Example 2 were used.

Infrared spectroscopy confirmed that PA had been reacted with said beadsthrough a Hofmann rearrangement.

EXAMPLE 14

The procedure of Example 8 was followed except that 100 ml ofdiethylamine (DEA), 3 ml of water and 2 g of the product of Example 2were used.

Infrared spectroscopy confirmed that DEA had been reacted with saidbeads through a Hofmann rearrangement.

EXAMPLE 15

The procedure of Example 8 was followed except that 1100 ml of3,3'-diamino-N-methylpropylamine, 28 ml of water, and 22 g of theproduct of Example 2 were used.

Infrared spectroscopy confirmed that 3,3'-diamino-N-methylpropylaminehad been reacted with said beads through a Hofmann rearrangement.

EXAMPLE 16

A mixture of 65 g of tris(hydroxymethyl)aminomethane (Tris) and 65 g ofwater was heated in an oil bath; the mixture turned to a clear solutionafter the temperature of the mixture reached 55° C. The solution cooledto 40° C., and to this solution was added 2 g of the product of Example2. The suspension was stirred at room temperature for two hrs. and thenfiltered. The beads were washed with water and vacuum dried (40° C.)

Infrared spectroscopy confirmed that tris(hydroxymethyl)aminomethane(Tris) had been reacted with said beads through a Hofmann rearrangement.

EXAMPLE 17

The procedure of Example 8 was followed except that 100 ml of3-diethylaminopropylamine, 2.0 ml of water and 2.5 g of the product fromExample 2 were used, and the reaction mixture was stirred at 40° C. for3 hours. Potentiometric titration of the beads using aq. hydrochloricacid revealed that the beads contained 0.86 meq/g of the amino group.

Infrared spectroscopy confirmed that diethylaminopropylamine had beenreacted with said beads through a Hofmann rearrangement.

EXAMPLE 18

To 260 ml of a 0.2N aq. sodium hydroxide solution was added 10 g of theproduct of Example 2. The suspension was stirred at room temperature for3 hrs. and then filtered. The beads were washed with water and vacuumdried (40° C.). Potentiometric titration using aq. hydrochloric acidshowed that the beads contained 0.33 meq/g of the carboxylic group.

EXAMPLE 19

A solution containing 50 g of dextran (mol. wt. 15,000) and 50 ml ofwater was heated to 40° C. To this solution was added 10 g of theproduct of Example 2 and 5 ml of 2N aq. sodium hydroxide solution. Thesuspension was stirred at 42° C. for 3 hrs. and then filtered. The beadswere washed with water and vacuum dried. Analysis using anthrone method(Anal. Chem. 25, 1656, (1953)) showed that the beads contained 3% by wt.dextran.

Infrared spectroscopy confirmed that said functional groups had beenreacted with said beads through a Hofmann arrangement.

EXAMPLE 20

The procedure of Example 8 was followed except that 130 ml ofpolyoxyethylenediamine (Jeffamine EDR-148), 3 ml of water and 5 g of theproduct of Example 2 were used.

Infrared spectroscopy confirmed that poloxyethylenediamine had beenreacted with said beads through a Hofmann rearrangement.

EXAMPLE 21

A solution of 117 ml of decylamine and 2.7 ml of water was heated to 40°C. To this solution was added 4.5 g of product of Example 2. Thesuspension was stirred at 40° C. for 2 hrs. and then filtered. The beadswere washed with acetone and hexanes and vacuum dried (40° C.).

Infrared spectroscopy confirmed that decylamine had been reacted withsaid beads through a Hofmann rearrangement.

EXAMPLE 22

A mixture of 10 g of octadecylamine and 15 ml of hexadecane was heatedin an oil bath. The mixture turned to a clear solution when thetemperature of the mixture reached 60° C. To this solution was added 0.6ml of water and 1.25 g of the product of Example 2. The mixture wasstirred at 62° C. for 75 minutes and then filtered. The beads werewashed with heptane and vacuum dried (40° C.).

Infrared spectroscopy confirmed that octadecylamine had been reactedwith said beads through a Hofmann rearrangement.

EXAMPLE 23

A solution of 75 ml of dioxane (dried over 3A molecular sieving) and10.5 g of carbonyldiimidazole was purged with nitrogen. The solution wasthen heated to 35° C., and to this solution was added 3.5 g of theproduct of Example 3. The suspension was stirred at 35° C. undernitrogen atmosphere for 1.5 hrs. and then filtered. The beads werewashed with acetone, cold water, THF and acetone and vacuum dried atroom temperature.

Infrared spectroscopy confirmed that carbonyldiimidazole had beenreacted with said beads.

EXAMPLE 24

A suspension of 10 g of ethylenediamine urea beads (the product ofExample 8), and 90 ml of 0.1N sodium chloride was immersed in a 4° C.ice bath. Powdered succinic anhydride (40 g) was slowly added withconstant stirring over 2 hrs. The pH was maintained at 6.0 with theaddition of 5N NaOH and the temperature was kept between 4° C. and 10°C. After the succinic anhydride addition, the temperature was maintainedat 4° C. and the pH was kept at 6.0 for an additional 4 hrs. The beadswere collected, washed with 1.0N hydrochloric acid, water, and methanol,and then vacuum dried. Titration results showed 145 micromoles ofcarboxyl groups/ml of beads.

EXAMPLE 25

Succinylated ethylenediamine urea beads (prepared as in Example 24), 2.0mls, containing 114 micromoles of carboxyl groups/ml, were dehydrated inp-dioxane. The beads were collected and added to 5 ml of dry p-dioxane.N-Hydroxysuccinimide, 500 micromoles, was added followed by 500micromoles of dicyclohexylcarbodiimide. Non-solvents for acrylonitrilepolymers or copolymers may comprise any liquid medium which isimmiscible therewith. These were tumbled overnight, collected, andwashed with dry p-dioxane and methanol. The activation density was 37micromoles/ml (determined by the method of T. Miron and M. Wilchek,Analytical Biochemistry, 126, 433-435 (1982)).

The following Examples illustrate the attachment of bioligands or otherfunctional groups to Substrate II through various bridging groups.

EXAMPLE 26

Succinylated ethylenediamine urea beads (prepared as in Example 24),0.25 g, containing 114 micromoles of carboxyl groups/ml, were added to2.5 ml of 0.1N NaCl. Ethylenediamene dihydrochloride, 0.45 g, was addedand the pH was adjusted to 4.7 with 0.1N NaOH1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDAC),0.049 was added, and this was tumbled 24 hrs. at room temperature whilemaintaining the pH at 4.7. The beads were collected, washed with 0.1NHCl, water, and methanol, and then vacuum dried. These beads contained98 moles amine/ml (determined by the method of G. Antoni, et al.,Analytical Biochemistry, 129, 60-63 (1983)).

EXAMPLE 27

The dehydrated product of Example 3, 10 mls, was added to 10 ml of dryacetonitrile containing 5500 micromoles of 4-dimethylaminopyridine(DMAP). 2-Fluoro-1-methyl-pyridinium toluene-4-sulfonate (FMP) 5,000micromoles, in dry acetonitrile, 25 ml, was added directly to the beadmixture. This was tumbled 2 hrs. at room temperature. The beads werewashed 1×100 ml acetonitrile, 2×100 ml acetone, and stored at 4° C. in30 ml of dry acetone. The beads contained 112 micromoles of activatedhydroxyl groups/ml, as assayed by the amount of 1-methyl-2-pyridonereleased when the beads were tumbled 24 hr. at room temperature in 0.2Nsodium hydroxide (see T. Ngo, Bio/Technology, Vol. 4, 134-137 (1986),for the spectrophotometric assay procedure).

The product produced was identified as a polymer containing1-methyl-2-pyridoxal toluene-4-sulfonate (MPTS) groups.

EXAMPLE 28

The procedure of Example 27 was followed except that 3.5 ml of theproduct of Example 12 was reacted with 1925 micromoles of DMAP in 3.5 mlof acetonitrile, and 1750 micromoles of FMP in 8.9 ml of acetonitrile.The density of activation was 27.3 micromoles/ml.

The product produced was identified as a polymer containing MPTS groups.

EXAMPLE 29

The procedure of Example 27 was followed except that 5.5 ml of theproduct of Example 50 was reacted with 3025 micromoles of DMAP in 5.5 mlof acetonitrile, and 2750 micromoles of FMP in 13.8 ml of acetonitrile.The density of activation was 15.5 micromoles/ml.

The product produced was identified as a polymer containing MPTS groups.

EXAMPLE 30

The procedure of Example 27 was followed except that 2.0 ml of theproduct of Example 16 was reacted with 1100 micromoles of DMAP in 2.0 mlof acetonitrile, and 1000 micromoles of FMP in 5.0 ml of acetonitrile.The density of activation was 18.8 micromoles/ml.

The product produced was identified as a polymer containing MPTS groups.

EXAMPLE 31

The procedure of Example 30 was followed except that a solution of 40%dimethylsulfoxide, 60% acetonitrile was used in place of acetonitrile.The density of activation was 28.5 micromoles/ml.

The product produced was identified as a polymer containing MPTS groups.

EXAMPLE 32

The procedure of Example 27 was followed except 10 ml of the product ofExample 7 was used. The density of activation was 23.7 micromoles/ml.

The product produced was identified as a polymer containing MPTS groups.

EXAMPLE 33

The procedure of Example 27 was followed except 10 ml of the product ofExample 5 was used. The density of activation was 70.1 micromoles/ml.

The product produced was identified as a polymer containing MPTS groups.

EXAMPLE 34

The dehydrated product of Example 3, 6 ml, was added to 18 ml of dryacetone. p-Nitrophenylchloroformate, 6000 micromoles, was added and themixture was immersed in a 4° C. ice bath. DMAP, 7200 micromoles in 10 mlof dry acetone, was added dropwise, and the contents were tumbled for 1hr. at 4° C. The beads were collected and washed with cold acetone, 5%acetic acid in dioxane, methanol, and isopropanol, and stored at 4° C.in isopropanol. The density of activation was 20.1 micromoles/ml(determined by the method of T. Miron and M. Wilchek, BiochemistryInternational, Vol. 4, No. 6, 629-635 (1982)).

The product produced was identified as a polymer containingp-nitrophenyl formate groups.

EXAMPLE 35

The dehydrated product of Example 3, 2.5 ml, was added to 4 ml of dryacetone containing 2500 micromoles of N,N'-disuccinimidyl carbonate. Asolution of 4250 micromoles of DMAP in 5 ml of acetone was slowly added.The suspension was tumbled for 1 hr. at 4° C. The beads were collectedand washed with cold acetone, 5% acetic acid in dioxane, methanol, andisopropanol. The activated beads were stored in 4° C. isopropanol. Thedensity of activation was 28.5 micromoles/ml (determined by the methodof M. Wilchek and T. Miron, Applied Biochemistry and Biotechnology, Vol.11, 191-193 (1985)).

The product produced was identified as a polymer containing activehydroxy succinimide carbonate (HSC) groups.

EXAMPLE 36

The procedure of Example 35 was followed except the product of Example 5was used. The density of activation was 25.6 micromoles/ml.

The product produced was identified as a polymer containing active HSCgroups.

EXAMPLE 37

The product of Example 27, 0.50 ml, was washed 3xlO ml water, 1×3 ml0.05N sodium carbonate/sodium bicarbonate, pH 8.5 (coupling buffer). Thecoupling buffer, 1.20 ml, containing 50 mg of bovine serum albumin (BSA)was added to the beads. After tumbling for 24 hrs. at room temperature,the beads were washed

with 4×10 ml 1.0N NaCl, and 3×10 ml water. The beads coupled 8.1 mgBSA/ml (determined from protein concentration in combined washings,using the Pierce BCA Protein Assay).

The product produced was identified as a polymer with immobilized BSA.

EXAMPLE 38

The procedure of Example 37 was followed except the product of Example28 was used. The beads coupled 5.8 mg BSA/ml.

The product produced was identified as a polymer with immobilized BSA.

EXAMPLE 39

The product of Example 35, 0.50 ml, was washed with 4° C. water and 4°C. 0.1N phosphate buffer, pH=7.5 (coupling buffer). Bovine serumalbumin, 50 mg, in 1.20 ml of coupling buffer was added. This wastumbled at 4° C. for 24 hrs. The beads were washed with 4x40 ml 1.0NNaCl, 3×10 ml water. The beads coupled 5.5 mg BSA/ml (determined fromthe protein concentration in the combined washings, using the Bio-RadProtein Assay).

EXAMPLE 40

The procedure of Example 37 was followed except that the product ofExample 33 (prepared with a lower density of activation of 16micromoles/ml), 2 ml was used. Cytochrome C, 12.2 mg, in 2.7 mls of 0.2NNaHCO₃ pH=9.0, was added to the beads. These beads coupled 1.5 mgCytochrome C/ml (determined from Bio-Rad Protein Assay).

EXAMPLE 41

The procedure of Example 40 was followed except the product of Example27 (prepared with a lower degree of activation of 25 micromoles/ml) wasused. The beads coupled 2.9 mg Cytochrome C/ml (determined by theBio-Rad Protein Assay).

The following Examples illustrate the practice of the present inventionwherein substrates other than beads are utilzed.

EXAMPLE 42

The product of Example 1B, 0.24 g, was mixed with 14 ml of water. Tothis mixture was added 0.066 g of chlorine in 4 minutes. After standingat room temperature for 1.5 hrs., the reaction mixture was filtered. Thefibers were washed with water and vacuum dried (40° C.) Iodometrictitration showed that the fibers contained 1.92 mmole/g of activechlorine.

EXAMPLE 43

A solution of 5 ml of diethylene glycol and 0.2 ml of 2N aqueous sodiumhydroxide solution was heated to 40° C. To this solution was added 0.12g of the product of Example 42. The reaction mixture was heated at 42°C. for 2 hrs. and then filtered. The fibers were washed with water andvacuum dried.

Infrared spectroscopy revealed that diethylene glycol had reacted withthe fibers by way of a Hofmann rearrangement.

EXAMPLE 44

The product of Example 1C, 0.41 g, was mixed with a solution of 20 ml ofwater and 0.10 g of chlorine. After standing at room temperature for 2hrs., the reaction mixture was decanted. The fiber was washed with waterand vacuum dried (40° C.). Iodometric titration showed that the fibercontained 0.70 mmole/g of active chlorine.

EXAMPLE 45

A solution of 15 ml of DEG and 0.4 ml of 2N aqueous sodium hydroxidesolution was heated to 40° C. To this solution was added 0.3 g of theproduct of Example 44. After standing at 40° C. for 3 hrs., the reactionmixture was decanted, and the fiber was washed with water and methanoland vacuum dried (40° C.).

Infrared spectroscopy revealed that diethylene glycol had reacted withthe fibers by way of a Hofmann rearrangement.

EXAMPLE 46

The procedure of Example 44 was followed and the product of Example 1D,0.38 g, was used for the reaction to produce a nonporous acrylonitrilecopolymer film having pendant N-chloroamide groups therefrom.

EXAMPLE 47

A mixture of 0.05 g of the product of Example 46, 10 ml of decylamine,and 0.2 ml of water was heated at 40° C. for 3 hrs. The reaction mixturewas then decanted. The film was washed with heptane several times andvacuum dried (room temperature). Water contact angle for the film was107°.

EXAMPLE 48

A mixture of 5 g of tri(hydroxymethyl)aminomethane and 5 g of water washeated to 55° C., and the mixture turned to a clear solution. Thesolution cooled to 50° C., and to this solution was added 0.12 g of theproduct of Example 46. After standing at 43° C. for 3 hrs., the reactionmixture was decanted. The film was washed with water and methanol andair dried. The water contact angle was 54°.

EXAMPLE 49

The procedure of Example 27 was followed except that 0.0585 g of theproduct of Example 43 was reacted with 320 micromole of DMAP in 0.582 mlof dry acetonitrile, and 290 micromoles of FMP in 1.450 ml of dryacetonitrile. The density of activation of the resulting product was241.0 micromole/g.

EXAMPLE 50

The procedure of Example 8 was followed except 26 ml ofN-methyldiethanolamine, 0.70 ml of water, and 1.0 g of the product ofExample 2 were used.

Infrared spectroscopy confirmed that the N-methyldiethanolamine hadreacted with the substrate through a Hofmann rearrangement.

EXAMPLE 51

The procedure of Example 8 was followed except that 3 g of the productof Example 2, 100 ml of N,N-diethylethylenediamine (DEAE) and 2 ml ofwater were used and the reaction mixture was stirred at 45° C. for 3hrs.

Infrared spectroscopy confirmed that DEAE had been reacted with saidbeads through a Hofmann rearrangement. Potentiometric titration usingaqueous hytrochloric acid showed that the beads contained 1.03 meq/g ofthe amino group.

EXAMPLE 52

A mixture of 52 g of glycine and 52 ml of 35% (w/w) aqueous sodiumhydroxide was heated to 35° C. and a clear solution was obtained. Thethis solution was added 4 g of the product of Example 2. The suspensionwas stirred at 40° C. for 3 hrs and then filtered. The beads were washedwith 0.1N aq. sodium hydroxide, and water and vacuum dried.

Infrared spectroscopy confirmed that glycerine had reacted with saidbeads through a Hofmann rearrangement Potentiometric titration showedthat the beads contained 1.20 meq/g of the carboxylate group.

EXAMPLE 53

The procedure of Example 52 was followed except that 3 g of the productof Example 2, 39 g of beta-alanine and 35 ml of 35% (w/w) aq. sodiumhydroxide were used.

Infrared spectroscopy confirmed that the beta-alanine had reacted withsaid beads through a Hofmann rearrangement. Potentiometric titrationshowed that the beads contained 1.23 meq/g of the carboxylate group.

EXAMPLE 54

The procedure of Example 52 was followed except that 3 g of the productof Example 2, 42 g of 6-aminocaproic acid, 23ml of 35% (w/w) aq. sodiumhydroxide were used, and before heating to 35° C., 16 ml of water wasadded to the mixture of 6-aminocaproic acid and 35% aq. sodiumhydroxide.

Infrared spectroscopy confirmed that the 6-aminocaproic acid had reactedwith said beads through a Hofmann arrangement. Potentiometric titrationshowed that the beads contained 1.09 meq/g of the carboxylate group.

EXAMPLE 55

The product of Example 45, 0.10 g, was washed 3×40 ml dry acetone, 5×40ml dry acetonitrile, and the solvent was removed. The fibrillated fiberswere added to 8 ml of dry acetonitrile containing 0.07 g of4-dimethylaminopyridine. 2-Fluoro-1-methyl-pryidiniump-toluene-sulfonate (FMP), 0.14 g, in 2.6 ml of dry acetonitrile wasadded all at once and the solution was tumbled for 2 hours at roomtemperature. The fibers were collected and washed 1×50 ml acetonitrile,and 2×100 ml acetone. The fibers contain 127 umoles of activatedhydroxyl groups/gram, when assayed with 0.2N sodium hydroxide (seeExample 27 for assay procedure). The fiber product was demonstrated tocontain MPTS groups.

The following Examples illustrate the use of various Biosubstratesproduced in accordance with the present invention.

EXAMPLE 56

The product of Example 55, 0.6 g, was washed 3×13 ml distilled water,and 1.13 ml 0.05N sodium carbonate, pH =8.5 (coupling buffer). Thebuffer was removed and 6.4 ml of coupling buffer containing 100 mg ofbovine serum albumin (BSA) was added. This was tumbled at roomtemperature for 3 hours, and then at 4° C. for 2 days. The fibers werewashed 3x100 ml of coupling buffer and 4×100 ml of 1.0N sodium chloride.The fibers coupled 4.44 mg BSA (7.3 mg BSA/g of fiber). This wasdetermined by performing the Pierce BCA*Protein Assay directly on thefiber.

EXAMPLE 57

The product of Example 27, 1 ml (prepared with 31 micromoles activatedhydroxyl groups/ml of bead), was washed 3×10 ml water, 1×10 ml 0.05Nsodium carbonate, pH =8.5 (coupling buffer). The coupling buffer wasremoved and 1 ml of degassed coupling buffer containing 8 mg of proteinA was added. This was tumbled 24 hours at room temperature and then 24hours at 4° C. The beads wee washed with 3 ml of 0.05N sodium chloride,and then a 75/25 methanol/water (v/v) solution over 1/2 hour. Afterwashing 1×10 ml 0.01 N sodium acetate, pH=4.5, the beads were stored at4° C. in 0.01N Tris-HCl, 0.1% sodium azide, pH=8.5. The beads were shownto have coupled Protein A.

EXAMPLE 58

The product of Example 55, 1.2 g, was reacted with 8 mg of Protein Afollowing the procedure of Example 57, except that a 5 fold solutionvolume was used to allow complete wetting of the fibers. The beads wereshown to have coupled Protein A.

EXAMPLE 59

The product of Example 57, (1 ml) was suspended in phosphate bufferedsaline (PBS), (0.10M sodium phosphate, 0.9% sodium chloride, 0.01%sodium azide, pH =7.4), and packed into a 5 ml chromatography column.The beads were washed with 5ml of a 0.1% acetic acid in 10% methanolbuffer (Regeneration Buffer) and then 15 ml of PBS (Binding Buffer), atflow rates of 40 ml/hr. Normal human serum (NHS) was diluted 1 part NHSto 2 parts binding buffer and filtered. 12 ml of the diluted NHS wasgravity fed through the column. After washing the beads with 20 ml fbinding buffer, purified IgG was eluted with a 0.1N glycine, pH=2.8buffer. Using the extinction coefficient at 280 nm of a 1% solution ofhuman IgG of 13.5, the binding capacity of the beads was shown to beabout 27.0 mg IgG/ml of beads.

EXAMPLE 60

The fibrillated fiber product (0.36 g) of Example 62, cut into three17/8 " circles, (0.36 g), and were packed into a cartridge (Millipore).The fibers were checked for IgG binding following the procedure ofExample 59. The binding capacity was shown to be about 9.1 mg IgG/gramof fibers.

EXAMPLE 61

The procedure of Example 3 was followed except that 15 grams of1,6-hexanediamine, 13.7 g of diethylene glycol, 1.0 ml of water, and 1.0g of the product of Example 2 were used. These beads contained 169micromoles of amine/ml of bead when determined by the method of Example26.

EXAMPLE 62

Following the procedure of H. J. Bohme, et al., (J. of Chromatography,69 (1972) 209-214), 0.4 g of Cibacron Blue F3G-A (Sigma) in 12 ml ofwater was added to 2 g of the product of Example 3 in 70 ml of 60° C.water. This was stirred for 1/2 hour and then 9 g of sodium chloride wasadded. This was heated to 80° C. and then 0.8 g of sodium carbonate wasadded. This was stirred for 2 hours, and then the beads were collectedand washed with water and methanol to give dark blue beads. It wasdetermined that the Cibacron Blue dye had been bonded to the beadsthrough a urethane-DEG linkage.

EXAMPLE 63

Underivatized fibrillated fiber, 0.5 g, was washed five times with 10 mlsolutions of 1.ON NaCl, DI water and 3×10 ml phosphate buffered saline(PBS), (0.01 M sodium phosphate, 0.9% sodium chloride, 0.01% sodiumazide, pH=7.4). PBS, 10 ml, containing 100 mg of BSA was contacted withthe fibers and this was tumbled at room temperature for 1 hour. Thefibers were washed with 4×10 ml distilled water and with 10×10 ml ofPBS. The assay for protein using the Pierce BCA* Protein Assay Reagentdirectly on the fiber showed 1.2 mg of BSA non-specifically bound to thefiber (2.4 mg BSA/gram of fiber).

EXAMPLE 64

The procedure of Example 64 was followed except 0.5 g of the product ofExample 45 was used. This fiber showed no non-specifically bound BSA.

EXAMPLE 65

A 1.13 g sample of spun acrylic yarn was treated as disclosed inExample 1. The examination of the surface by ESCA revealed the presenceof amide groups.

EXAMPLE 66

The product of Example 27, 1.0 ml, prepared with 33 micromoles ofactivated hydroxyl groups/ml, was coupled with BSA following theprocedure of Example 37, except that 25 mg of BSA was added/ml of beads.The amount of coupling was 3.5 mg BSA/ml of beads.

EXAMPLE 67

The procedure of Example 66 was repeated, except that a 40% Ethanol, 60%coupling buffer (v/v), was used. The amount of coupling was 13.3 mgBSA/ml of beads.

EXAMPLE 68

The procedure of Example 2 wasw followed except that a reduced amount ofchlorine gas was used to produce a product with 0.27 meq ofN-chloroamide per gram of product. A solution of 37.5 grams of malonicdihydrazide and 82.5 grams of water to 45 degrees C and stirred untilthe dihydrazide dissolved. Triethylamine, 0.31 grams and 7.5 grams ofthe above N-chloroamide product were added and allowed to stir for twohours at 45 degrees C. The product was then collected on a Buchnerfunnel and washed with warm water, cold water, 0.1 N HCl and cold water.Colorimetric assay showed 20 microequivalents of hydrazide bound per mLof resin.

EXAMPLE 69

The product of Example 7 was tumbled for 3 hours in a solution of 0.001N sodium periodate in 0.1 N acetate buffer, pH 5.0. The reaction wasfollowed by disappearance of the periodate. The product was collectedand washed with 0.05 N acetate buffer (pH 5.0) to give beads with analdehyde functional surface.

EXAMPLE 70

The product of Example 69 was added to a solution of 0.001 N adipicdihydrazide in 0.05 N acetate buffer (pH 5.0). The suspension wastumbled for 4 days at room temperature, collected, and washed withwater. The TNBS hydrazide assay showed the presence of about 12micromoles of hydrazide per mL of beads.

EXAMPLE 71

A mixture of 62 grams of 2-aminoethane sulfonic acid, 36 mL of 35% byweight of aqueous sodium hydroxide and 20 mL of water was heated to 50degrees C until a clear solution was obtained. The solution was cooledto 43 degrees C and 5 grams of the product of Example 2 was addedthereto. Stirring was continued for 3 hours at 40 degrees C. The productwas collected and washed with water, 0.5 N sodium hydroxide, water,methanol and then air dried. Infrared spectroscopy confirmed thepresence of sulfonic acid groups, potentiometric titration showed 0.96meq per gram of product.

EXAMPLE 72

To a solution of 10 grams of ethyl chloride in 70 mL of methanol isadded 3 grams of the product of Example 51. The suspension is stirred atroom temperature for 3 hours. The product is then collected and washedwith water. Analysis reveals the presence of a quaternary ammoniumfunctional surface.

EXAMPLE 73

The procedure of Example 37 was followed except that fetuin was usedinstead of BSA, two different FMP activation loadings were used and thecoupling reaction was conducted for about 40 hours. The Pierce BCAProtein Assay was then conducted to show that the beads respectivelypossessed 44 and 123 micromoles of activated hydroxyl groups per ml and3.9 and 7.0 mg of bound fetuin per ml.

We claim:
 1. A process for the preparation of surface-modified beadsuseful in isolation of biological material, said process comprising:a)contacting beads comprising polyacrylonitrile, or a copolymer ofacrylonitrile and at least one comonomer, with an alkaline catalyst, aperoxide, and optionally a reducing agent under reaction conditions andfor a time sufficient to convert at least a portion of the nitrilegroups distributed on the surface of the substrate to amide groups; b)reacting said beads with a halogenating reagent under conditions and fora time sufficient to convert at least a portion of the amide groups toN-haloamide groups; c) reacting said beads with bioactive ligandselected from the group consisting of carboxylic acids, sulfonic acids,teritary amines, quaternary amines, peptides, hormones, enzymecofactors, enzyme substrates, enzyme inhibitors, antigens, antibodies,dyes, pigments, complex metal ions, proteins, nucleic acids,p-aminobenzamides, polysaccharides, lectins, non-proteinaceous toxins,and antiotoxins, under conditions and for a time sufficient to effectthe bonding of said ligand to said beads through said N-haloamide group;and d) recovering the resultant surface-modified beads.
 2. The processof claim 1 wherein said substance comprises a copolymer ofpolyacrylonitrile wherein the polyacrylonitrile content of saidsubstrate ranges from about 19 to 99 parts by weight.
 3. The process ofclaim 2 wherein the polyacrylonitrile content of said substrate rangesfrom about 50 to about 98 parts by weight.
 4. The process of claim 1wherein said comonomer is selected from the group consisting of C₂ -C₆mono-olefins, vinyl aminoaromatics, alkenyl aromatics, vinyl aromatics,vinyl halides, C₂ -C₆ alkyl(meth)acrylates, acrylamides,methacrylamides, vinyl pyrrolidones, vinyl pyridine, C₁ -C₆ hydroxestersof alkyl(meth)acrylates, meth(acrylic)acids, acrylomethylpropylsulfonicacids, N-hydroxy-containing C₁ -C₆ alky(meth)acrylamide,acrylamidomethylpropylsulfonic acids vinyl acetate, glycidyl(meth)acrylate, glycerol (meth)acrylate, tris(hydroxymethyl)aminomethyl(meth)acrylamide and mixtures thereof.
 5. The process of claim 1 whereinsaid substrate comprises a copolymer of acylonitrile and methyl acrylatewherein said acrylonitrile comprises at least 90 mole percent of saidsubstrate.
 6. The process of claim 1 wherein said N-haloamide groups areselected from the group consisting of N-chloroamide, N-iodoamide andN-bromoamide groups.
 7. The process of claim 1 wherein said N-haloamidegroups comprise N-chloroamide groups.
 8. The process of claim 1 whereinsaid bond of said bioligand and said substrate further comprises abridging group.
 9. The process of claim 8 wherein said bridging group isselected from the group consisting of C₁ to C₁₅ aliphatic, aromatic,cycloaliphatic groups which optionally contain heteroatoms, such as O, Nor S.
 10. The process of claim 1 wherein said bridging group is selectedfrom the group consisting of ethylene glycol, diethylene glycol,triethylene glycol, glycerol, ethylenediamine, diethylenetriamine,ethanolamine, diethanolamine, 3,3'-diamino-N-methylpropylamine,hexanediamine, glycine, beta-alanine, tris(hydroxymethyl)aminoethane,6-aminocaproic acid and polyoxyethylenediamine.
 11. The product producedby the process of claim
 1. 12. A composition of matter comprising:aporous bead comprising polyacrylonitrile or a copolymer of acrylonitrileand at least one comonomer, and b) a surface having evenly distributedthereon,i) pendant bioactive ligand groups selected from the groupconsisting of carboxylic acids, sulfonic acids, tertiary amines,quaternary amines, peptides, hormones, enzyme cofactors, enzymesubstrates, enzyme inhibitors, antigens, antibodies, dyes, pigments,complex metal ions, proteins, nucleic acids, p-aminobenzamides,polysaccharides, lectins, non-proteinaceous toxins, and antiotoxins saidbioactive ligand groups being bound to said surface through linkagesderived from the reaction of bioactive ligands and N-haloamide groupsbound to said surface, and, optionally ii) nitrile and/or amide groups.13. The composition of claim 12 said bead comprises a copolymer ofpolyacrylonitrile wherein the polyacrylonitrile content of saidsubstrate ranges from about 29 to 99 parts by weight.
 14. Thecomposition of claim 13 wherein the polyacrylonitrile content of saidsubstrate ranges from about 50 to about 98 parts by weight.
 15. Thecomposition of claim 12 wherein said comonomer is selected from thegroup consisting of C₂ -C₆ mono-olefins, vinyl aminoaromatics, alkenylaromatics, vinyl aromatics, vinyl halides, C₂ -C₆ alkyl(meth)acrylates,acrylamides, methacrylamides, vinyl pyrrolidones, vinyl pyridine, C₁ -C₆hydroxesters of alkyl(meth)acrylates, meth(acrylic)acids,acrylomethylpropylsulfonic acids, N-hydroxy-containing C₁ -C₆alky(meth)acrylamide, acrylamidomethylpropylsulfonic acids vinylacetate, glycidyl (meth)acrylate, glycerol (meth)acrylate,tris(hydroxymethyl)aminomethyl (meth)acrylamide and mixtures thereof.16. The composition of claim 12 wherein said bead comprises a copolymerof acrylonitrile and methyl acrylate wherein said acrylonitrilecomprises at least 90 mole percent of said copolymer.
 17. Thecomposition of claim 12 wherein said N-haloamide groups are selectedfrom the group consisting of N-chloroamide, N-iodoamide and N-bromoamidegroups.
 18. The composition of claim 12 wherein said N-haloamide groupscomprise N-chloroamide and N-bromoamide groups.
 19. The composition ofclaim 12 wherein said bond of said bioligand and said substrate furthercomprises a bridging group.
 20. The composition of claim 19 wherein saidbridging group is selected from the group consisting of C₁ to C₁₅aliphatic, aromatic, cycloaliphatic groups which optionally containheteroatoms, such as O, N or S.
 21. The process of claim 12 wherein saidbridging group is selected from the group consisting of ethylene glycol,diethylene glycol, triethylene glycol, glycerol, ethylenediamine,diethylenetriamine, ethanolamine, diethanolamine,3,3'-diamino-N-methylpropylamine, hexanediamine, glycine, beta-alanine,tris(hydroxymethyl)aminomethane, 6-aminocaproic acid andpolyoxyethylenediamine.